title: Crypt_KO_S10
description: Protocols: Colon intestinal crypts were isolated following previously published protocol (Sato 2013). Briefly, the entire colon was excised, flushed with cold PBS and opened longitudinally. Small pieces of colon (2-4 mm in length) were cut with scissors and further washed with ice-cold PBS until the supernatant was clear. Next, tissue fragments were incubated in cold 2 mmol/L EDTA chelation buffer, for 1 h at 4 °C. After removal of the EDTA buffer, tissue fragments were vigorously resuspended in cold chelation buffer using a 10-mL pipette-BSA coated to isolate intestinal crypts. The tissue fragments were allowed to settle down under normal gravity for 1 minute, and the supernatant was removed for inspection by inverted microscopy. The resuspension/sedimentation procedure was repeated typically 8 times and the supernatants containing crypts (from wash 1 to 8) were collected in 50-mL Falcon tubes coated with BSA. Isolated crypts were pelleted, washed in PBS, and centrifuged at 200g for 3 minutes at 4 °C to separate crypts from single cells. Crypts were resuspended in 10 ml basal crypt medium (BCM, advanced Dulbecco's modified Eagle medium -F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM Glutamax) to quantify its number. Colon tissue was used for muscle isolation. After flushing with cold PBS, colons were open in longitudinal and dissected under a bench scope. Using a coverslip, mucosal tissue was completely removed carefully under the dissection scope and muscle was deep frozen in N2 for RNA extraction. References Sato and Clevers. 2013. Primary Mouse Small Intestinal Epithelial Cell Cultures. Epithelial Cell Culture Protocols: Second Edition Tissues were first placed in lysis buffer (RLT, provided in RNeasy® Mini Quiagen Kit, 74104) and disrupted with sterile ceramic beads (Magna Lyser green beads tubes 03358941001) using a Tissue Lyser (FastPrep-24™, SKU 116004500), with two rounds of 6500 rpm for 30 seconds each, with care taken to maintain the sample cold. RNA isolation was performed following manufacturer instructions (RNeasy® Mini Kit, Qiagen, 74104). Library preparation and sequencing was performed by NTNU Genomic Core facility. Lexogen SENSE mRNA lib prep kit was used to generate the library.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-05-01T00:23:34Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-03T19:10:20Z
attributes.tag: External Id
attributes.value: SAMEA6872406
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:34Z
attributes.tag: INSDC last update
attributes.value: 2020-06-03T19:10:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9180:Crypt_KO_S10
attributes.tag: age
attributes.value: 8 to 12
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: Mmp17-/-
attributes.tag: individual
attributes.value: A
attributes.tag: organism part
attributes.value: epithelium of crypt of Lieberkuhn
attributes.tag: sample name
attributes.value: E-MTAB-9180:Crypt_KO_S10
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6